1. Field of the Invention
The present invention relates to a method for characterizing signals generated from molecular events at the single molecule level, such as donor-acceptor fluorescent resonance energy transfer events, of dynamic systems or static systems over a period of time, where the event data can be collected continuously, periodically, or intermittently and analyzed continuously, periodically or intermittently. The data collection and analysis, thus, can be in real time or near real time, while analysis can be any time post collection. A dynamic system means that the data is collected on the system in real time over the period of time as the system undergoes detectable changes in one or more detectable properties, while a static system means that the data is collected for a given period of time and the system is unchanging during that period of time.
More particularly, the present invention relates to a method for characterizing signals generated from detectable molecular events at single molecule level, where the method includes the steps of collecting and storing data from a viewing field associated with a detector, where the viewing field includes a plurality of molecules or molecular assemblies capable of being detected directly and undergoing a detectable event or a plurality of detectable events, where direct detection involves monitoring at least one detectable property associated with the molecule or molecular assembly and where the detectable events involve interactions associated with or occurring at the molecule or molecular assembly. Data associated with the viewing field is collected into one data channel or a plurality of data channels, where each data channel corresponds to an attribute of the detected events, such as intensity, frequency or wavelength, duration, phase, attenuation, etc. The method also includes the step of reading the stored data and spatially registering or calibrating the data channels so that a given location within the viewing field in one channel corresponds to the same location in the other channels—the data is registered relative to the viewing field. After registering, candidate molecules or molecular assemblies are identified. The candidate identification is generally designed to minimize locations within the viewing field that include more than a single directly detected molecule or molecular assembly to simplify data analysis. Next, an n×m array of data elements such as pixels is selected for each candidate so that the array includes all data elements having a detection value above a definable threshold originating from or associated with each candidate such as a definable intensity threshold value. Then, a plurality of “dark” data elements or pixels in an immediate neighborhood of the array associated with each candidate are selected to improve background removal. Once the array and background elements have been selected, a hybrid dataset for each candidate is constructed derived from data from two or more data channels. The hybrid dataset is then smoothed and differentiated. After smoothing and differentiating, non-productive events are separated from productive events based on a set of criteria, where the criteria are dependent on the detectable property and events being detected. The productive events are then placed in time sequence. For donor-acceptor systems, the method includes determining anti-correlated donor and acceptor fluorescent signals. For monomer sequencing (nucleotide, amino acid, saccharide, etc.), the criteria are designed to separate binding and mis-incorporation events from true incorporation events, and when placed in time order, evidence a sequence of monomers in a target sequence of monomers.
2. Description of the Related Art
With the increase in single molecular analytical techniques, there have been developed many software routines for analyzing the resulting data. However, each single molecule analytic technique gives rise to many unique problems and normal analytical software is ill suited to analyze data from very specific single molecule data detection systems.
Thus, there is a need in the art for data processing processes that can help researchers understand and characterize data corresponding to detectable events arising at the single molecule level especially in the area of single molecule fluorescence detection such as fluorescent resonance energy transfer signals originating from interactions between a donor or plurality of donors and an acceptor or a plurality of acceptors.